PROJECT SUMMARY Staphylococcus aureus is a significant cause of morbidity and mortality that is increasingly acquiring resistance to all available antibiotics. One promising area for antimicrobial development involves targeting bacterial acquisition and utilization of nutrient metal. This strategy exploits the fact that all bacterial pathogens require nutrient metal to colonize their hosts. In response to this requirement, vertebrates have evolved powerful defense strategies that sequester nutrient metals from invading pathogens in a process known as nutritional immunity. One of the most effective metal chelating factors of the immune system is calprotectin, an abundant protein that sequesters nutrient manganese, iron, and zinc and defends against microbial infection. In addition to its metal chelating properties, calprotectin is a potent pro-inflammatory molecule. The individual importance of each of these activities to protection against infection has not been parsed, and the contribution of calprotectin metal binding to its immunomodulatory properties is not known. Prevailing models suggest that nutritional immunity ensures that pathogens are uniformly metal starved during vertebrate colonization. We have challenged this concept through the application of innovative imaging technologies, revealing S. aureus is differentially metal starved within abscesses. This discovery necessitates a reevaluation of the environment encountered by S. aureus during infection, particularly as it pertains to nutrient metal levels. We have also discovered that the S. aureus response to metal restriction includes the up-regulation of members of the newly described COG0523 family of zinc metallochaperones. Our preliminary results reveal that these enzymes enable S. aureus to combat calprotectin-mediated zinc depletion by delivering zinc to critical metalloproteins involved in genome maintenance. Based on these fundamental discoveries, we hypothesize that following colonization of the vertebrate host, S. aureus encounters immune-mediated metal restriction through the delivery of calprotectin to the site of infection. In response, S. aureus up-regulates the expression of systems to combat this nutrient limitation. We envision that this microbial response is heterogeneous, and occurs in a manner dependent on the level of metal restriction experienced at distinct sites of infection. Finally, we predict that an important aspect of this response to nutrient limitation is the up-regulation of dedicated metallochaperones that deliver metal to proteins involved in DNA repair and are required for survival upon exposure to the reactive oxygen burst of the phagocyte. Experiments in this proposal will test this model by (i) defining the heterogeneous S. aureus response to metal starvation, (ii) elucidating the mechanism by which calprotectin protects against infection, and (iii) determining the subcellular fate of metal acquired by S. aureus during metal deprivation. This work will provide a mechanistic framework for understanding the importance of nutritional immunity during bacterial infection and reveal the corresponding S. aureus response to this host defense.